Methods and compositions for RNA interference
The present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA). The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).
1. A population of mammalian cells stably transfected or stably transduced with a library of expression constructs, wherein each expression construct encodes a short hairpin RNA, each expression construct comprising:
(i) an RNA polymerase promoter, and
(ii) a sequence encoding a short hairpin RNA molecule comprising a double-stranded region wherein the double-stranded region consists of at least 20 nucleotides but not more than 29 nucleotides,
wherein the short hairpin RNA molecule is a substrate for Dicer-dependent cleavage and does not trigger a protein kinase RNA-activated (PKR) response in the mammalian cells,
wherein the double-stranded region of the short hairpin RNA molecule comprises a sequence that is complementary to a portion of a target gene in the mammalian cells, wherein the target gene is an endogenous gene of the mammalian cells, and
wherein said population of mammalian cells comprises cells in which the short hairpin RNA molecule is stably expressed in an amount sufficient to attenuate expression of the target gene in a sequence specific manner without use of a PKR inhibitor.
2. The population of mammalian cells of claim 1 , wherein the expression construct further comprises LTR sequences located 5′ and 3′ of the sequence encoding the short hairpin RNA molecule.
3. The population of mammalian cells of claim 1 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of at least 21 nucleotides.
4. The population of mammalian cells of claim 1 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of at least 22 nucleotides.
5. The population of mammalian cells of claim 1 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of at least 25 nucleotides.
6. The population of mammalian cells of claim 1 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of 29 nucleotides.
7. The population of mammalian cells of claim 1 , wherein the short hairpin RNA molecule has a total length of 70 nucleotides.
8. The population of mammalian cells of claim 1 , wherein the RNA polymerase promoter comprises a pol II promoter or a pol III promoter.
9. The population of mammalian cells of claim 8 , wherein the pol III promoter comprises a U6, an HI, or an SRP promoter.
10. The population of mammalian cells of claim 8 , wherein the pol II promoter comprises a Ul or a CMV promoter.
11. A mammalian cell stably transfected or stably transduced with an expression construct encoding a short hairpin RNA molecule, the expression construct comprising:
(i) an RNA polymerase promoter, and
(ii) a sequence encoding a short hairpin RNA molecule comprising a double-stranded region wherein the double-stranded region consists of at least 20 nucleotides but not more than 29 nucleotides,
wherein the short hairpin RNA molecule is a substrate for Dicer-dependent cleavage and does not trigger a protein kinase RNA-activated (PKR) response in the mammalian cell,
wherein the double-stranded region of the short hairpin RNA molecule comprises a sequence that is complementary to a portion of a target gene in the mammalian cell, wherein the target gene is an endogenous gene of the mammalian cell, and
wherein the short hairpin RNA is stably expressed in an amount sufficient to attenuate expression of the target gene in a sequence specific manner without use of a PKR inhibitor.
12. The mammalian cell of claim 11 , wherein the expression construct further comprises LTR sequences located 5′ and 3′ of the sequence encoding the short hairpin RNA molecule.
13. The mammalian cell of claim 11 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of at least 21 nucleotides.
14. The mammalian cell of claim 11 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of at least 22 nucleotides.
15. The mammalian cell of claim 11 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of at least 25 nucleotides.
16. The mammalian cell of claim 11 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of 29 nucleotides.
17. The mammalian cell of claim 11 , wherein the short hairpin RNA molecule has a total length of 70 nucleotides.
18. The mammalian cell of claim 11 , wherein the RNA polymerase promoter comprises a pol II promoter or a pol III promoter.
19. The mammalian cell of claim 18 , wherein the pol III promoter comprises a U6, an HI, or an SRP promoter.
20. The mammalian cell of claim 18 , wherein the pol II promoter comprises a Ul or a CMV promoter.